Regulation of N-methyl- d-aspartate receptor expression and N-methyl- d-aspartate-induced cellular response during chronic hypoxia in differentiated rat PC12 cells

The purpose of the present study was to examine the effect of chronic hypoxia on N-methyl- d-aspartate-mediated cellular responses in differentiated PC12 cells. PC12 cells were differentiated by treatment with nerve growth factor. Patch-clamp analysis in differentiated PC12 cells showed that extracellularly applied N-methyl- d-aspartate induced an inward current that was abolished by the presence of the N-methyl- d-aspartate receptor antagonist MK-801. Results from Ca 2+ imaging experiments showed that N-methyl- d-aspartate induced an elevation in intracellular free Ca 2+ which was also abolished by MK-801. We also examined the effect of hypoxia on the N-methyl- d-aspartate-induced current in nerve growth factor-treated cells. We found that the N-methyl- d-aspartate-induced inward current and the N-methyl- d-aspartate-induced elevation in intracellular free Ca 2+ were markedly attenuated by chronic hypoxia. We next examined the possibility that the reduced N-methyl- d-aspartate responsiveness was due to down-regulation of N-methyl- d-aspartate receptor levels. Northern blot and immunoblot analyses showed that both messenger RNA and protein levels for N-methyl- d-aspartate receptor subunit 1 were markedly decreased during hypoxia. However, the messenger RNA for N-methyl- d-aspartate receptor subunit 2C was increased, whereas the protein level for subunit 2C did not change. Our results indicate that differentiated PC12 cells express functional N-methyl- d-aspartate receptors and that chronic exposure to hypoxia attenuates the N-methyl- d-aspartate-induced Ca 2+ accumulation in these cells via down-regulation of N-methyl- d-aspartate receptor subunit 1. This mechanism may play an important role in protecting PC12 cells against hypoxic stress.